A multi-parameter approach to measurement of spontaneous myogenic contractions in human stomach: Utilization to assess potential modulators of myogenic contractions.

Electrical slow waves, generated by interstitial cells of Cajal (ICC), cause spontaneous contractions of human stomach. Software was developed to measure muscle tone and eleven different parameters defining these contractions in human stomach, displaying data as radar plots. A pilot study assessed the effects of potential modulators, selected from among compounds known to influence ICC activity; n = 4-7 each concentration tested/compound. Human distal stomach (corpus-antrum) muscle strips were suspended in tissue baths for measuring myogenic (non-neuronal) contractions in the presence of tetrodotoxin (10-6 M). Initial characterization: Contractions (amplitude 4 ± 0.4mN, frequency 3 ± 0.1 min-1, n = 49) were unchanged by ꭃ-conotoxin GVIA (10-7 M) or indomethacin (10-6 M) but abolished by nifedipine (10-4 M). Carbachol (10-7 M) increased contraction rate and amplitude; 10-6-10-5 M increased tone and caused large, irregular contractions. [Ca2+]imodulators: Ryanodine (10-5-10-4 M) increased muscle tone accompanied by inhibition of myogenic contractions. Xestospongin-C (10-6 M; IP3 channel inhibitor) had no effects. SERCA pump inhibitors, 2-APB and cycloplazonic acid (10-5-10-4 M) increased tone and myogenic contraction amplitude before abolishing contractions; thapsigargin was weakly active. CaCC blockers: MONNA and CaCCinh-A01 had little-or-no effects on tone but reduced myogenic contractions; MONNA (10-4 M) was more effective, reducing amplitude (77.8 ± 15.2%) and frequency. CaV3.1/3.2/3.3 channel block: Mibefradil reduced tone and myogenic contraction amplitude (pIC50 4.8 ± 0.9). Inward-rectifying K+-channel inhibitor: E-4031 (10-4 M) increased contraction duration (17.4 ± 5.8%). Conclusions: (1) Measurement of multiple parameters of myogenic contractions identified subtle differences between compounds, (2) only E-4031 and CaCC blockers influenced myogenic contractions, not muscle tone, (3) studies are needed with compounds with known and/or improved selectivity/potency for human targets affecting ICC functions.

Total collagen content and distribution is increased in human colon during advancing age.

BACKGROUND: The effect of ageing on total collagen content of human colon has been poorly investigated. The aim of this study was to determine if ageing altered total collagen content and distribution in the human colon. METHODS: Macroscopically normal ascending colon was obtained at surgery from cancer patients (n = 31) without diagnosis of diverticular disease or inflammatory bowel disease. Masson's trichrome and Picrosirius red stains were employed to identify the total collagen content and distribution within the sublayers of the colonic wall for adult (22-60 years; 6 males, 6 females) and elderly (70 - 91years; 6 males, 4 female) patients. A hydroxyproline assay evaluated the total collagen concentration for adult (30-64 years; 9 male, 6 female) and elderly (66-91 years; 8 male, 8 female) patients. KEY RESULTS: Histological studies showed that the percentage mean intensity of total collagen staining in the mucosa, submucosa and muscularis externa was, respectively, 14(1.9) %, 74(3.2) % and 12(1.5) % in the adult ascending colon. Compared with the adults, the total collagen fibres content was increased in the submucosa (mean intensity; 163.1 ± 11.1 vs. 124.5 ± 7.8; P < 0.05) and muscularis externa (42.5 ± 8.0 vs. 20.6 ± 2.8; P < 0.01) of the elderly patients. There was no change in collagen content of the mucosa. The total collagen concentration was increased in the elderly by 16%. Sex-related differences were not found, and data were combined for analysis. CONCLUSIONS: Greater total collagen content was found in the submucosa and muscularis externa of the elderly human male and female colon. These changes may contribute to a possible loss of function with ageing.

Synergistic augmentation of rhythmic myogenic contractions of human stomach by arginine vasopressin and adrenaline: Implications for the induction of nausea.

BACKGROUND AND PURPOSE: Nausea is associated with the hormonal secretion of vasopressin and adrenaline, although their actions in inducing nausea is poorly understood. Here, we have investigated their actions on human stomach muscle. EXPERIMENTAL APPROACH: Muscle strips were suspended in tissue baths and neuronal-/non-neuronally-mediated contractions were measured. Custom software analysed eight motility parameters defining spontaneous phasic non-neuronally mediated contractions. Receptor distributions were assessed by qPCR and immunofluorescence. KEY RESULTS: V1A receptors and α1 -adrenoceptors were located on muscle as well as interstitial cells of Cajal (ICCs). Myogenic contractions of human proximal and distal stomach (respectively, 2.6 ± 0.1 and 2.7 ± 0.0 per minute; n = 44) were larger in the distal area (1.1 ± 0.1 and 5.0 ± 0.1 mN), developing relatively slowly (proximal) or rapidly (distal). Vasopressin caused tonic (proximal) or short-lived (distal) increases in muscle tone and increased myogenic contraction amplitude, frequency and rate (acting at V1A receptors; thresholds 10-11 -10-10  M); by contrast, cholinergically mediated contractions were unaffected. Oxytocin acted similarly to vasopressin but less potently, at OT receptors). Adrenaline increased (10-10 -10-5  M; α1 -adrenoceptors) and decreased (≥10-6  M; ß-adrenoceptors) muscle tone and enhanced/reduced myogenic contractions. Cholinergically mediated contractions were reduced (α2 -adrenoceptors). Combined, vasopressin (10-9  M) and adrenaline (10-8  M) increased muscle tone and phasic myogenic activity in a synergistic manner. CONCLUSIONS AND IMPLICATIONS: Vasopressin and adrenaline increased human gastric tone and myogenic contraction amplitude, rate of contraction and frequency. In combination, their actions were further increased in a synergistic manner. Such activity may promote nausea.

Expression of p16 Within Myenteric Neurons of the Aged Colon: A Potential Marker of Declining Function.

Human colonic neuromuscular functions decline among the elderly. The aim was to explore the involvement of senescence. A preliminary PCR study looked for age-dependent differences in expression of CDKN1A (encoding the senescence-related p21 protein) and CDKN2A (encoding p16 and p14) in human ascending and descending colon (without mucosa) from 39 (approximately 50: 50 male: female) adult (aged 27-60 years) and elderly donors (70-89 years). Other genes from different aging pathways (e.g., inflammation, oxidative stress, autophagy) and cell-types (e.g., neurons, neuron axonal transport) were also examined. Unlike CDKN1A, CDKN2A (using primers for p16 and p14 but not when using p14-specific primers) was upregulated in both regions of colon. Compared with the number of genes appearing to upregulate in association with temporal age, more genes positively associated with increased CDKN2A expression (respectively, 16 and five of 44 genes studied for ascending and descending colon). Confirmation of increased expression of CDKN2A was sought by immunostaining for p16 in the myenteric plexus of colon from 52 patients, using a semi-automated software protocol. The results showed increased staining not within the glial cells (S100 stained), but in the cytoplasm of myenteric nerve cell bodies (MAP2 stained, with identified nucleus) of ascending, but not descending colon of the elderly, and not in the cell nucleus of either region or age group (5,710 neurons analyzed: n = 12-14 for each group). It was concluded that increased p16 staining within the cytoplasm of myenteric nerve cell bodies of elderly ascending (but not descending) colon, suggests a region-dependent, post-mitotic cellular senescence-like activity, perhaps involved with aging of enteric neurons within the colon.